The invention relates to biological analysis, and more particularly, to a method of determining the presence of and measuring the amounts of the co-enzymes NADH and NADPH.
As is known, the aforementioned abbreviations denote the following substances:
NADH=reduced form of NAD (or NAD+) (nicotin-amide-adenine dinucleotide). also called co-enzyme I or DPN (diphosphopyridine nucleotide) PA0 NADPH=reduced form of NADP (or NADP+) (nicotinamide-adenine-dinucleotide phosphate), also called co-enzyme II or TPN (triphosphopyridine nucleotide).
The reversible conversion of NAD into NADH is shown diagrammatically as follows (where R denotes a ribose-diphosphate-ribose-adenine chain): ##STR1##
The aforementioned enzymes play a part in a large number of biochemical enzymatic reactions used as clinical tests. One example of these reactions is oxidation of .alpha.-hydroxyacids into corresponding ketonic acids in the presence of a suitable dehydrogenase. One example is the oxidation of lactic acid or lactates to pyruvic acid, which is represented as follows: ##STR2## Similarly. glucose-6-phosphate is oxidized in the presence of NADP and glucose-6-phosphate dehydrogenase (G6PDH) to glucoma-.delta.-lactone-6-phosphate and NADPH. The determination of NADP or NADH in an aforementioned reaction is very important, since it can be indirectly used for determining glucose in biological fluids after they have been converted into glucose-6-phosphate in the presence of ATP (adenosine triphosphate) and hexokinase (HK).
NADH also acts as a co-enzymatic factor in the conversion of 2-oxoglutarate into L-glutamate by ammonium salts in the presence of GLDH (glutamate dehydrogenase), thus enabling the ammonium in the reaction medium to be determined by measuring the NADH.sup.+ formed. This can be used for determining urea in biological fluids, since urea in the presence of urease supplies NH.sub.3 which occurs as the NH.sup.+.sub.4 ion in the aforementioned conversion.
Similarly, the determination of transaminase in blood serum which catalyzes the conversion of .alpha.-ketoglutarate into oxaloacetate (SGOT=serum glutamate oxaloacetate transaminase) is very important in clinical chemistry, since an excess of this enzyme can indicate a coronary thrombosis.
Measurement of SGOT is associated with measurement of the reduction of the oxaloacetate formed during the aforementioned reaction by NADH in the presence of malate dehydrogenase (MDH) or glutamate dehydrogenase (GLDH). The reaction diagram is as follows: ##STR3##
A description of other applications associated with the determination of the NAD.sup.+ and NADH factors are found in the following documents: EP-A-29 104 (MILES); FR-A-2 299 644 (AKZO).
In view of the importance of determining the aforementioned co-enzymes in one or the other of their states of oxydo-reduction, numerous techniques have been proposed for this purpose.
For example, since NAD and NADH have different absorptions in the UV range of the spectrum, one form can be determined in the presence of the other by spectrophotometry. The sensitivity of spectrophotometric determination can also be amplified by combined use of colored redox compounds, for example tetrazolium compounds which form intensely colored formazan salts in the presence of NADH or NADPH and an electron acceptor such as phenazine methosulphate (See, for example, document EP-A-114 267). Alternatively, use may be made of fluorimetric techniques as described, for example, in document FR-A-2 266 644.
Alternatively, an electrochemical method may be used as described in document JP 56 035 50. where NADH or NADPH is oxidized with Meldola blue, after which the reduced form of the dye is electrochemically oxidized and the oxidation current is measured.
The following reaction has recently been recommended (see R. H. WHITE-STEVENS et al.. J. Biol. Chem. (1972) 247. 2358; EP-A-29 104): ##STR4##
The thus-liberated hydrogen peroxide (H.sub.2 O.sub.2) is then determined by conventional means, for example, by its action, catalyzed by peroxydase, on a redox indicator, the oxidized form of which is determined by colorimetry.
This technique is very attractive but is of use only in a colorless, optically transparent medium, which is far from being the case with most biological fluids used for analysis. Also, in operation this technique requires the presence of a "coupler" for preventing the dyed oxidized compound from being reversibly reduced by NADH in the analysis medium. In order to determine H.sub.2 O.sub.2 under these conditions, it is therefore desirable to have a technique supplying identifiable products in an irreversible manner. With regard to these techniques, it has recently been disclosed (see EP-A-20 623) that excellent results with regard both to sensitivity and accuracy can be obtained in the analysis of H.sub.2 O.sub.2 produced by oxidation of glucose in the presence of glucose oxidase, the method involving reacting the H.sub.2 O.sub.2 with an aromatic fluorinated compound in the presence of peroxidase so as to liberate fluoride ions, which are then electrometrically determined by using an electrode specific to these ions.